Aminopyrimidine derivatives as antiviral agents for respiratory syncytial virus

ABSTRACT

Antiviral activity against respiratory syncytial virus has been found in some substituted 6-aminopyrimidines having the formula: ##STR1## wherein R 1  is lower alkyl, preferably methyl or t-butyl, R 2  is halogen or cyano, and R 3  is C 1  -C 6  alkyl or --(CH 2 ) n  R 4  where n is 1 or 2 and R 4  is phenyl, phenyl substituted by lower alkoxy, lower alkyleneoxy, bromo, or 3,4-methylenedioxy; 3 or 4-pyridinyl, pyridinyl substituted by cyano or bromo, thienyl, or di-(C 1  -C 6  alkyl)amino.

FIELD OF INVENTION

This invention relates to antiviral activity found in some 6-substituted amino-4-pyrimidinone compounds. Belshe and Hay, J. Respiratory Diseases 10:552-561, 1989, hypothesize that the M2 protein of influenza virus and the SH protein of respiratory syncytial virus (RSV) may function as ion channels. The compounds used in the method of this invention have certain structural similarities to the known potassium channel opener, pinacidil (N-cyano-N'-4-pyridinyl-N"-1,2,2-trimethylpropylguanidine). Two of the compounds which had good antiviral activity against RSV were tested against influenza A and found to be inactive against the influenza A virus.

Respiratory syncytial virus (RSV) is a pneumovirus that causes respiratory infections. RSV is the single most frequent cause of serious respiratory disease in young children, often resulting in lower respiratory tract infections. (Volck, et al, Essentials of Medical Microbiology, 3rd ed., 1986, J. B. Lippincott Co., Philadelphia, p. 727). RSV infections are currently treated with ribavirin, a synthetic nucleotide that is administered intranasally as an aerosol.

DESCRIPTION OF THE INVENTION

The compounds useful in the method of this invention were synthesized for cardiovascular activity and are disclosed in U.S. Pat. Nos. 4,505,910; 4,617,393; 5,002,949 and in J. Med. Chem. 1988, 31, 814-823 and are herein incorporated by reference. The compounds are represented by Formula I below: ##STR2## wherein: R¹ is C₁ -C₆ lower alkyl;

R² is halogen or --CN;

R³ is C₁ -C₆ alkyl or --(CH₂)_(n) R⁴ where n is 1 or 2 and R⁴ is phenyl, phenyl substituted by 1 to 2 groups selected from lower alkoxy, lower alkenyloxy, halogen or 3,4-methylenedioxy;

pyridinyl, substituted pyridinyl where the substituent is selected from halogen or cyano, thienyl, or --NR¹ R² where R¹ and R² are C₁ -C₆ alkyl or --NR¹ R² is a cyclic amine of 4 to 6 atoms;

or a pharmaceutically acceptable salt thereof.

By way of further definition of terms used above, C₁ -C₆ alkyl includes straight and branched chain hydrocarbons, lower alkoxy is --O--(C₁ -C₆ alkyl), lower alkenyloxy is --O--(C₁ -C₆ alkenyl) where the alkenyl group may have one site of unsaturation, halogen is selected from fluorine, chlorine, bromine or iodine, and --NR¹ R² defined as a cyclic amine of 4 to 6 atoms is selected from azetidine, pyrrolidine or piperidine.

The term pharmaceutically acceptable salt includes solvates, hydrates, acid addition salts and quaternary salts. The acid addition salts are formed from a Formula I compound having a basic nitrogen and a pharmaceutically acceptable inorganic or organic acid including but not limited to hydrochloric, hydrobromic, sulfuric, phosphoric, methanesulfonic, acetic, citric, malonic, fumeric, maleic, sulfamic, or tartaric acids. Quaternary salts are formed from a basic Formula I compound and an alkyl or arylalkyl halide, preferably methyl or benzyl bromide.

Preferred Formula I for use in this invention are those wherein R¹ is methyl or t-butyl, R² is bromo, chloro, fluoro or cyano, R³ is 2-methylbutyl or --(CH₂)_(n) R⁴ and R⁴ is phenyl, 3-methoxy-4-(2-propenyloxy)phenyl, 5-bromo-2-methoxyphenyl, 3-thienyl, 3-pyridinyl, 4-pyridinyl, 3-thienyl, 3-pyridinyl, 4-pyridinyl, 5-bromo-3-pyridinyl, 5-cyano-3-pyridinyl, 3,4-methylenedioxyphenyl, dimethylamino, dipropylamino, or 1-pyrrolidinyl or a pharmaceutically acceptable salt thereof.

More preferred compounds for use in this invention are those wherein R¹ is t-butyl, R² is --CN and R³ is 3-pyridinylmethyl or when R¹ is methyl and R² is --CN, then R³ is selected from 2-(3-pyridinyl)ethyl, 2-(4-pyridinyl)ethyl, phenylmethyl, 2-dimethylaminoethyl, or 2-(1-pyrrolidinyl)ethyl; and when R¹ is methyl and R² is Br, R³ is selected from 5-cyano-3-pyridinylmethyl, 2-(3-pyridinyl)ethyl, 2-dipropylaminoethyl, 3,4-methylenedioxyphenylmethyl, 3-thienylmethyl, 2-[3-methoxy-4-(2-propenyloxy)-phenyl]ethyl, 2-(5-bromo-2-methoxyphenyl)ethyl, 2-methylbutyl, or 5-bromo-3-pyridinylmethyl; or when R¹ is methyl and R² is Cl, R³ is 5-bromo-3-pyridinylmethyl or a pharmaceutically acceptable salt thereof.

The compounds useful in this invention exist as tautomers in either the keto (Ia or Ib) or enol (Ic) forms as shown below, depending on the chemical environment. The keto structure Ia is used therein. ##STR3##

The useful compounds are summarized in the Table 1 below.

                  TABLE 1     ______________________________________     Summary of structures of Anti-RSV Formula I Compounds     Com-     pound R.sup.1                  R.sup.2                         R.sup.3     ______________________________________     1     CH.sub.3                  CN                          ##STR4##     2     CH.sub.3                  Br                          ##STR5##     3     t-Bu   CN                          ##STR6##     4     CH.sub.3                  CN                          ##STR7##     5     CH.sub.3                  CN                          ##STR8##     6     CH.sub.3                  CN     CH.sub.2 CH.sub.2 N(CH.sub.3).sub.2     7     CH.sub.3                  CN                          ##STR9##     8     CH.sub.3                  Br                          ##STR10##     9     CH.sub.3                  Br     CH.sub.2 CH.sub.2 N(CH.sub.2 CH.sub.2 CH.sub.3).sub.2                         7     10    CH.sub.3                  Br                          ##STR11##     11    CH.sub.3                  Br                          ##STR12##     12    CH.sub.3                  Br                          ##STR13##     13    CH.sub.3                  Cl                          ##STR14##     14    CH.sub.3                  Br                          ##STR15##     15    CH.sub.3                  F                          ##STR16##     16    CH.sub.3                  Br     CH.sub.2 CH.sub.2 N(CH.sub.3).sub.2     17    CH.sub.3                  Br                          ##STR17##     ______________________________________

DETAILED DESCRIPTION OF THE INVENTION

The antiviral activity against respiratory syncytial virus was determined in HEp-2 cells seeded in 96 well culture plates. The compounds (0.16-16 μg/ml) were added to the cells 2 hours prior to RSVA₂ infection (˜500 PFU/well) and the plates were stained with crystal violet 3 days after infection to reveal the cytopathic effects. Antiviral activities were determined in triplicate, while cellular toxicities were determined in a single test. The anti-flu activities of Compounds 1 and 2 against FluA/WSN were similarly determined in Madin Darby Canine Kidney (MDCK) cells except ˜25 PFU of the virus were used and the plates were stained 2 days after infection. Compounds 1 and 2 were inactive against FluA/WSN.

The test data for anti-RSV are shown in Table 2.

                  TABLE 2     ______________________________________     Formula I Compound Anti-RSV Test Data                  Anti-RSVA.sub.2                             Cellular Toxicity     Compound     ED.sub.50, μg/ml                             TD.sub.50, μg/ml     ______________________________________     1            0.05       >16     2            0.05       >16     3            1.6-5      >16     4            0.16       >16     5            1.6        >16     6            5          >16     7            1.6         16     8            0.005      .sup. >16.sup.a     9            1.6        >16     10           (0.05).sup.b                             >16     11           0.5        >16     12           0.5        >16     13           (0.016).sup.c                             >16     14           0.5        >16     15           (0.05).sup.b                             >16     16           (0.16).sup.b                             >16     17           (0.016).sup.c                             >16     Ribavirin    1.6-5       50     Amantadine   50          100     ______________________________________      .sup.a 20% cytotoxicity observed at ≧0.016 μg/ml      .sup.b No more than 50% protection at higher dose      .sup.c No more than 80% protection at higher dose

The compounds of Examples 1 and 2 were active against both subgroup A (RSVA₂) and subgroup B (RSV-9320) of RSV in HEp-2 cells with an ED₅₀ 10- to 30-fold less than that of ribavirin and 100- to 300-fold less than that of amantadine. The results are shown in Table 3.

                  TABLE 3     ______________________________________     Comparison of Examples 1 and 2, Amantadine and Ribavirin for     Anti-RSV Activity in HEp-2 Cells             Median Inhibitory Concentration (IC.sub.50, μg/ml)             Respiratory Syncytial Virus     Compound          RSVA.sub.2                                RSV-9320     ______________________________________     Ex. 1             ≦0.16                                ≦0.16     Ex. 2             ≦0.16                                ≦0.16     Amantadine        50       16     Ribavirin         1.6-5    5     ______________________________________

The cytotoxic activities for the compounds of Examples 1 and 2, amantadine and ribavirin were determined in five different cell lines. The test results are summarized in Table 4.

                  TABLE 4     ______________________________________     Comparison of Invention Compounds, Amantadine and Ribavirin     for Cellular Toxicity             Minimal Toxic Dose (μg/ml)             that Inhibits All Growth     Compound  MDCK     HEp-2    A549  MRC-5 Vero     ______________________________________     Ex. 1     >100     >100     >100  100   >100     Ex. 2.sup.a                >50      50       >50  >50   >50     Amantadine               >100     100      >100  100   100     Ribavirin  100      50       100  100   100     ______________________________________      .sup.a Not completely soluble at 100 μg/ml

Based on the in vitro anti-respiratory syncytial virus test data of the invention compounds as compared with ribavirin, the contemplated dosage of a Formula I compound for intranasal or intrabronchial administration is 10 to 30 times less than that of ribavirin. Oral or parenteral dosage forms may be given in one dose or divided doses. The exact dosage will, of course, be determined according to standard medical principles by a physician, beginning with a low dose and increasing the dosage until the desired antiviral effect is obtained.

The compounds may be administered neat or with a pharmaceutical carrier to a mammal in need thereof. The pharmaceutical carrier may be solid or liquid.

A solid carrier can include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material. In powders, the carrier is a finely divided solid which is in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredients. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.

Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For parenteral administration, the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in sterile liquid form compositions for parenteral administration. The liquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.

For administration by intranasal or intrabronchial inhalation or insufflation, compounds of this invention can be formulated into an aqueous or partly aqueous solution, which can then be utilized in the form of an aerosol.

Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administrated intravenously. The compound can also be administered orally either in liquid or solid composition form. 

What is claimed is:
 1. A method of treating viral infections in mammals caused by respiratory syncytial virus which comprises administration to a mammal in need thereof a therapeutically effective amount of a compound of the formula: ##STR18## wherein: R¹ is C₁ -C₆ lower alkyl;R² is halogen or cyano; R³ is C₁ -C₆ alkyl or --(CH₂)_(n) R⁴ where n is 1 or 2 and R⁴ is phenyl, phenyl substituted by 1 to 2 groups selected from lower alkoxy, lower alkenyloxy, halogen, and methylenedioxy; pyridinyl, pyridinyl substituted by halogen or cyano, thienyl, or --NR¹ R² when R¹ and R² are C₁ -C₆ alkyl or --NR¹ R² is a cyclic amine of 4-6 atoms; or a pharmaceutically acceptable salt thereof.
 2. The method according to claim 1 where, in the compound used, R¹ is methyl and R² is bromo, chloro, fluoro or cyano or a pharmaceutically acceptable salt thereof.
 3. The method according to claim 2 where, in the compound used, R¹ is methyl and R² is bromo.
 4. The method according to claim 3 where, in the compound used, R³ is selected from 5-cyano-3-pyridinylmethyl, 2-(3-pyridinyl)ethyl, 2-(dipropylamino)ethyl, 3,4-methylenedioxyphenylmethyl, 3-thienylmethyl, 2-[3-methoxy-4-(2-propenyloxy)phenyl]ethyl, 2(5-bromo-2-methoxyphenyl)ethyl, 3-methylbutyl, and 5-bromo-3-pyridinylmethyl or a pharmaceutically acceptable salt thereof.
 5. The method according to claim 2 where, in the compound used, R² is cyano.
 6. The method according to claim 5 where, in the compound used, R³ is selected from 2-(3-pyridinyl)ethyl, 3-pyridinylmethyl, 2-(4-pyridinyl)ethyl, phenylmethyl, 2-(dimethylamino)ethyl, and 2-(1-pyrrolidinyl)ethyl or a pharmaceutically acceptable salt thereof.
 7. The method according to claim 2 wherein the compound used is 6-[[(5-bromo-3-pyridinyl)methyl]amino]-5-chloro-2-methyl-4(1H)-pyrimidone or a pharmaceutically acceptable salt thereof.
 8. The method according to claim 2 wherein the compound used is a 5-fluoro-2-methyl-6-[(3-pyridinylmethyl)amino]-4(1H)-pyrimidone or a pharmaceutically acceptable salt thereof.
 9. The method according to claim 1 where, in the compound used, R¹ is t-butyl.
 10. The method according to claim 9 wherein the compound used is 5-cyano-2-(1,1-dimethylethyl)-6-[(3-pyridinylmethyl)amino]-4(1H)-pyrimidone or a pharmaceutically acceptable salt thereof. 